The Discreet Charm of the Curve

Antimicrobial peptides are found in a large variety of organisms throughout all branches of cellular life: bacteria, archaea, and eukarya. They provide an evolutionarily ancient mechanism of defense against microbial invasion and play a fundamental role in the innate immunity of animals and humans. The initial discovery of Cecropin peptides in silk moth pupae, where they are expressed in response to insect injury and infection and during development (1), and the subsequent isolation of Magainin peptides from the skin of the African clawed frog, Xenopus laevis (2), established their importance as potent antibiotics (3). Early electrophysiological studies showed that Magainins, and other related peptides, cause highly selective and rapid lysis of bacteria in a concentration-dependent manner. The peptides are capable of forming pores that rapidly disrupt the electrical potential across membranes of bacterial cells, isolated mitochondria, and synthetic vesicles (3). Furthermore, the biological activities of antimicrobial peptides are highly synergistic; strong synergism is observed in their antimicrobial activity as well as in their cytotoxicity for other cell types, including transformed tumor cells (3). The finding that antimicrobial peptides synthesized from all D amino acids retain their full biological activity provided the first evidence that their function does not involve interaction of the peptide with a membrane receptor but instead relies on interaction of the peptide with the lipid bilayer membrane (4). This is consistent with the highly positively charged amphiphilic structures common to all peptides, which confer specificity for the predominantly electronegative membranes of bacteria. However, the functional mechanism of antimicrobial peptide activity has been a subject of intense discussion. In this issue, Strandberg et al. (5) describe the subtle effects of phospholipid structure on the interactions of antimicrobial peptides with membranes. Their results provide an explanation for the peptides' selective cytotoxicity for bacteria. Strandberg et al. (5) demonstrate that the interactions of both individual and synergized antimicrobial peptides with membranes depend not only on the electrostatic attraction between a cationic peptide and the anionic membrane surface, but also, very significantly, on the fundamental chemical structures of the phospholipids that influence their predisposition for membrane curvature. Negative curvature predisposition is observed in lipids with unsaturated hydrocarbon chains, while more ordered, saturated chains typically result in positive curvature (Fig. 1). Figure 1 Antimicrobial peptide (AMP) surface association is promoted by lipids with negative curvature predisposition. Lipids are shown with anionic headgroups (red) and hydrocarbon chains (yellow). The amphiphilic helical peptide, viewed down its helix axis, ... While, in some cases, antimicrobial peptides can insert across lipid bilayer membranes, their predominant conformation as membrane surface-associated α-helices was demonstrated early on by solid-state NMR structural studies (6,7). Membrane surface association has been elegantly rationalized as an essential early step in the functional mechanism of antimicrobial peptides, leading to massive pore formation and bacterial cell death. Rather than forming a classic channel with peptide helices inserted across the lipid bilayer membrane, the accumulation of peptides at the membrane surface is thought to cause a strain in the lipid bilayer, resulting in the formation of toroidal pores where the essential alignment of the peptide helices relative to the geometry of individual lipids is maintained (8–10). In a series of elegant solid-state NMR experiments Strandberg et al. (5) probe the membrane-associated conformations of two synergistic Magainin peptides, MAG2 and PGLa, as a function of phospholipid bilayer composition. The data unequivocally show that the membrane-surface-associated states of both isolated and combined MAG2 and PGLa are stabilized by phospholipids whose structures predispose them to negative membrane curvature. MAG2 and PGLa do not insert in lipid bilayers composed of negative curvature lipids with unsaturated hydrocarbon chains, regardless of the hydrocarbon chain length. By contrast, phospholipids with fully saturated dimyristoyl and dipalmitoyl hydrocarbon chains and a predisposition for positive curvature allow transmembrane insertion of PGLa and antagonize the surface-bound state. Notably, such lipids also fail to elicit appreciable membrane depolarization by the peptides (3), suggesting that the membrane inserted state may not be critical for function. The results obtained for membranes composed of bacterial phospholipids are particularly revealing. In the presence of bacterial lipids, with palmitoyl and oleoyl hydrocarbon chains and cardiolipin, phosphoethanolamine, and phospho-glycerol headgroups, both MAG2 and PGLa helices always align parallel to the membrane surface. These lipids have a marked propensity for negative membrane curvature and are enriched in bacterial membranes against which Magainin peptides exhibit their highest biological activity. Thus, the data suggest that membrane surface association of the peptides, promoted by negative curvature lipids, is a key aspect of their antimicrobial cytotoxicity. Recently, cardiolipin microdomains have been shown to localize to negatively curved regions of Escherichia coli membranes (11) and a clear phenomenological link has been demonstrated between anionic lipid clustering and the bacterial species specificity of several antimicrobial agents (12). The results of Strandberg et al. (5) help explain how lipid clustering could result in bacterial toxicity: the anionic and negative curvature properties of bacterial lipid clusters could conspire to recruit the accumulation of antimicrobial peptides and maintain them in a membrane surface orientation up to the critical concentration beyond which membrane disruption and massive pore formation are inevitable and commit the cell to death. Interestingly, the mitochondrial trans-acylating enzyme Tafazzin was recently shown to interact with specific pools of lipids that possess negative curvature properties (13). Just as proposed for Tafazzin, the data of Strandberg et al. (5) do not demonstrate that the peptides interact with curved membrane regions but, instead, how they interact with lipids that have a natural tendency to induce negative curvature in membranes. Antimicrobial peptides have been the focus of a large number of biophysical and biological studies aimed at understanding the molecular basis for their activity. Now, Strandberg et al. (5) provide significant new insights that can advance their development as effective therapeutics.